Interpretive Summary: Foot-and-Mouth Disease Virus (FMDV) is one of the most contagious agents of cloven-hoofed species. Current vaccines are efficacious but require at least 7 days to induce protection against disease. Therefore, there is a need to develop countermeasure strategies that effectively work prior to this elapsed time. We have previously reported that mutation of FMDV leader protein SAP motif [SAP-mutant] results in attenuated growth in laboratory cell culture. Here, we studied the ability of the SAP-mutant to cause disease in swine. In a first experiment, we evaluated the virulence of this virus by inoculating different doses of SAP-mutant or wild type (WT) virus and looking at the response for 21 days. Animals inoculated with the SAP-mutant did not develop clinical signs, did not have virus infection in the blood stream or shedding from nasal and oral cavities even when the inoculation dose was 100-fold higher than that needed to cause disease by the wild-type virus. Since the induction of neutralizing antibodies was equivalent for both viruses, we evaluated the SAP mutant as a candidate vaccine against challenge with WT virus. Animals vaccinated with the SAP mutant were completely protected after inoculated with WT FMDV at 21 days post vaccination. We also performed a second experiment, challenging with WT FMDV at different times post vaccination (2, 4, 7 and 14 days). Remarkably, animals inoculated with the SAP-mutant virus were protected against challenge with WT-FMDV as early as 2 days post-vaccination before antibodies were detectable. In addition, animals inoculated with SAP- mutant developed a memory cellular immune response resembling infection with WT-FMDV. Our results suggest that SAP-mutant, or similar viruses containing mutations in the leader protein, may serve as the basis for effective live attenuated FMD vaccines.

Technical Abstract:
Foot-and-mouth disease virus (FMDV) leader proteinase (L^pro) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L^pro is also associated with degradation of nuclear factor kappaBeta(NF-kappaBeta), a process that requires L^pro nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L^pro coding sequence, SAP mutation, prevented L^pro nuclear retention and degradation of NF-kappaBeta, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP mutant virus does not cause clinical signs of disease, viremia or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild type (WT) virus. Remarkably, SAP inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 and for at least 21 days post inoculation. Early protection correlated with a distinct pattern in the serum levels of pro-inflammatory cytokines in comparison to animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L^pro plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L^pro coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against FMD.